Mitochondria provides energy for cells via oxidative phosphorylation (OXPHOS). The average cell contains hundreds to thousands of maternally-inherited mitochondria replete with their own mitochondrially-encoded genome. The interactions of mitochondrial and nuclear-encoded OXPHOS genes are critical in energy generation. Mutations in both nuclear- and mitochondrially-encoded OXPHOS genes have been identified but few targeted animal models exist. Defects within the OXPHOS genes are intimately associated with various metabolic diseases including severe childhood disorders including Leigh disease. Leigh disease is a neurodegenerative mitochondrial disorder characterized by a motor control loss and localized necrotic lesions. Leigh Disease can result from either mitochondrial or nuclear mutations-that cause devastating muscle and neurological degeneration between 3 months and 2 years of age along with a cascade of debilitating pathologies. This project focuses on developing genetically-engineered mouse models to characterize the role of specific OXPHOS genes and their involvement in OXPHOS pathways and disease progression. The majority of Leigh disease cases, whether acquired or sporadic, result from an insufficiency in production or function of particular electron transport proteins involved in oxidative phosphorylation. Various mutations in the NADH dehydrogenase-ubiquinone-FeS 4 (NDUFS4) gene have been associated with decreased Complex I activity in mitochondrial electron transport and Leigh disease. Specific phosphorylation of the NDUFS4 protein occurs in response to cAMP-dependent kinase activation and correlates with activation of Complex I. Conditional Cre-LoxP NDUFS4 heterozygous knockout mouse embryonic stem cells will be created to evaluate in vitro phenotype and to subsequently establish a homozygous knockout mouse model to examine in vivo phenotype and tissue specificity. A second embryonic stem cell line will be constructed to contain a heterozygous NDUFS4 point mutation. This will facilitate the creation of a homozygous point mutant mouse model that will address the specific mechanisms by which loss of a functional NDUFS4 protein can lead to Leigh disease. Phenotype will be identified by gross and histopathological analyses, in addition to specific measures of Complex I activity, cellular respiration and mitochondrial function. The 2 NDUFS4 mouse models will allow examination of mechanisms leading to Leigh disease. These models will further our understanding of mitochondrial function and Complex I assembly. As such, this project represents a first step toward developing therapeutic strategies and targeted interventions for debilitating mitochondrial disorders. [unreadable] [unreadable]